This invention relates to a protein which inhibits bacterial DNA gyrase activity (DGI) and to a process for the preparation thereof. This invention further relates to a method for screening and identifying an antimicrobial agent and the like, by assaying the action of compounds on the activity or expression of DGI. Furthermore, this invention relates to an antisense DNA (or RNA) of a gene which encodes DGI and an antibody to DGI.
Since penicillin had been discovered, various antibiotics have been used for treating microbe infections and have a great contribution to medical treatments. Major antibiotics which have been used presently in clinical treatments are xcex2-lactam antibiotics and new quinolone antibiotics. The action and mechanism of new quinolone antibiotics are found to be the inhibition of bacterial DNA gyrase.
Bacterial DNA gyrase is an essential enzyme for replication and proliferation of bacteria, since the gyrase introduces a negative supercoiled structure into chromosomal DNA, or unravels and separates tangled daughter DNA just after completion of replication, and it is constituted of two subunits (A and B).
As DNA gyrase inhibitors, compounds such as novobiocin and cyclothiazine are known in addition to new quinolone antibiotics, but proteinous inhibitors have not been known.
Regarding the control of DNA gyrase activity, Horiuchi et al. have reported that LetD and LetA proteins encoded on the F factor which is a plasmid of Escherichia coli are involved in inactivation of DNA gyrase and reactivation of inactive gyrase, respectively (Journal of Biological Chemistry, vol. 267, pp. 12244-12251, 1992). However, inactivation with LetD protein is found only in experiments using the whole of microbial cells, and the inactivation action is not recognized in in vitro reconstitution experiments. Further, there is no report on DNA gyrase-inhibiting proteins encoded on a bacterial chromosome.
Recently, the appearance of bacteria tolerant against the conventional xcex2-lactam or new quinolone antibiotics becomes a serious problem and antibiotics with new action and mechanism which have efficacy to these tolerant bacteria have been desired.
An object of the present invention is to provide a DNA gyrase-inhibiting protein (DGI) useful for the creation of antibiotics with new action and mechanism and a process for preparing the same. Further, another object is to provide a method for screening and identifying an antimicrobial agent with new action and mechanism in which the activity of DGI and expression thereof are modulated using DGI or a gene therefor.
The present inventors have found that, when the supercoiling activity of each fraction is measured and each fraction is analyzed with SDS-polyacrylamide electrophoresis in the course of carrying out the purification of Escherichia coli DNA gyrase using novobiocin-affinity column chromatography, there are present fractions which contain a holoenzyme of DNA gyrase, but they do not show the supercoiling activity and these fractions contain a protein with about 18 kDa in common. The present inventors have found the absolutely new finding that the about 18 kDa protein has the action of inhibiting the DNA gyrase activity (supercoiling activity), and named the 18 kDa protein xe2x80x9cDNA gyrase-inhibiting proteinxe2x80x9d (DGI) (also referred to as GyrI). Further, the present inventors have cloned a gene encoding this protein (dgi gene) (also referred to as gyrI gene) from the chromosome of Escherichia coli to establish a process for preparing DGI. The present inventors have further constructed a system for measuring DGI activity using purified DGI, and a system for measuring expression of DGI using a promoter region of the dgi gene. Furthermore, the present inventors have found that the growth of bacteria can be controlled by modulating the activity or expression of DGI and thereby accomplished the invention.
The present invention relates to a protein which inhibits the activity of bacterial DNA gyrase (DNA gyrase-inhibiting protein; DGI).
Further, the present invention relates to a host cell transformed by a replicable expression vector which contains a DGI-encoding DNA, and to a process for preparing DGI comprising culturing the host cell.
The present invention also relates to a process for identifying a medical compound comprising assaying the action of modulating DNA gyrase-inhibiting activity that DGI has, and to a process for identifying a medical compound characterized by assaying the action of modulating the expression of a DGI-encoding gene.
Furthermore, the present invention relates to an antisense DNA or an antisense RNA of a DGI-encoding gene, and to a DGI-recognizing antibody.